Telomeres are essential components of terminal chromosome structure that protect chromosome ends from degradation and recombination, and prevent chromosome loss. While telomeres cannot be fully replicated by conventional polymerases, the specialized RNA-dependent polymerase telomerase can maintain telomeric DNA. By evolutionarily conserved mechanisms, telomere length is determined by a tightly regulated equilibrium between this telomerase addition and degradative processes. In organisms as diverse as baker's yeast and mice, telomerase preferentially elongates short telomeres. The goals of this grant are to determine how telomeric DNA length regulates telomerase by using yeast as a model system. It is believed that Rap1p, the yeast telomeric DNA binding protein, is "counted" by the cell to monitor telomere length. Two Rap1-interacting factors, Rif1p and Rif2p, are thought to act immediately downstream of Rap1p. Deletion of either RIF gene disrupts the telomere length equilibrium in favor of lengthening, while double deletion of RIF1 and RIF2 has a synergistic effect on telomere length. In yeast, telomerase is also regulated temporally, with activity being limited to late S-phase. Recent evidence has suggested that Est1p, while dispensable for in vitro activity, may act as an activator of telomerase in vivo, and thus may target telomerase activity to short telomeres in S-phase. This proposal is designed to investigate the roles of Est1p, Rif1p, and Rif2p in telomere length regulation in yeast.